Molec. Gen. Genetics 106, 263--273 (1970)

Mechanism of Conjugation and Recombination in Bacteria I X . The Role of D N A Synthesis in Post-conjugal Genetic R e c o m b i n a t i o n ~¢[IROSLAWAWLODARCZYKand WLADYSLAWKUI~ICKI-GOLDF1-NGEt¢ Department of General Microbiology, Institute of Microbiology, University of Warsaw, Warsaw, Poland

Received December 8, 1969 Summary. Development of resistance to 23p-decay of donor genetic determinants after their transfer into the female cell is dependent on unabated DNA synthesis. A similar dependence upon DNA synthesis was found in recombinational events. Both processes show a similar time-course. The DNA synthesis, involved, seems distinct from physiological replication of the chromosome. The formation of the structure resistant to a2p-deeay is going on concomitantly with recombinational process and is completed within 45 to 55 minutes after transfer, before beginning of the replication of recombinant structure. The bearing of these facts on the molecular mechanism of genetic recombination is discussed.

Introduction An investigation of the type, amount and timing of DNA synthesis concomitant with genetic recombination should provide a clue to the molecular mechanism of recombination. Evidence from experiments on rec- m u t a n t s of Escherichia coli (Clark and Margulies, 1965; Howard-Flanders and Theriot, 1966) points to the role of a repair mechanism. The relationship between the repair-mechanism and the genetic recombination seems, however, more complicated than it had been assumed originally. Investigations of Clark and Barbour (1969) on enzymic make-up of rec- mutants, and of Piekarowicz (1969) on a fate of the donor DNA in the rec- recipient, indicate that, in this case, some preparatory step is blocked, which is necessary for recombinational events sensu stricto. The bearing of these facts on the molecular mechanism of genetic recombination is discussed. The results of Pritehard (1965) and Joshi and Siddiqi (1968) suggest t h a t normal, physiological replication of DNA is iness6ntial in post-conjugal recombinational events. Contradictory observations were reported by Bressler, Lanzov and Lukjaniee-Blinkova (1968). This discrepancy is hard to explain without additional experiments and controls. There is little, and very often controversial, information about the relationship between DNA synthesis and recombination in Euearyota (Hotta, I t o and Stern, 1966; Abel, 1967; Grell, 1967; Chiang and Sueoka, 1967; Esposito, 1968; Westergaard and Wettstein, 1968). Moreover, hypotheses on the molecular mechanism of recombination in Eucaryota (Holliday, 1964; Whitehouse and Hastings, 1965; Whitehouse, 1966, 1967; Fogel and Hurst, 1967) have to be modified to account for some processes, specific for proearyotic organisms only. Among these, the most important seem to be, the single-strandedness of

264

M. Wiodarczyk and W. Kunicki-Goldfinger:

t h e d o n o r ' s D N A ( K u n i c k i - G o l d f i n g e r , P i e k a r o w i c z a n d W t o d a r c z y k , 1968; P i e k a r o w i c z a n d K n n i c k i - G o l d f i n g e r , 1968; Cohen, l~isher, Curtiss a n d Adler, 1968; K u n i c k i - G o l d f i n g e r , 1969; P i e k a r o w i c z , 1969), t h e d e v e l o p m e n t of resistance t o 32p-decay of t h e d o n o r ' s genetic d e t e r m i n a n t s a f t e r t r a n s f e r to t h e F - c e l l ( J a c o b a n d W o l l m a n , 1961) a n d t h e occurrence of m a n y r o u n d s of r e c o m b i n a t i o n in successive g e n e r a t i o n s of t h e e x c o n j u g a n t cell (Lederberg, 1957; A n d e r s o n , 1958; Bressler, L a n z o v a n d B l i n k o v a , 1967; W o o d , 1967). T h e p r e s e n t s t u d y was designed to see w h e t h e r D N A s y n t h e s i s p l a y s a n y role in t h e f o r m a t i o n of r e c o m b i n a n t s t r u c t u r e s a n d in t h e d e v e l o p m e n t of resistance t o 82P-decay. I t h a s b e e n a s s u m e d t h a t , t h e role of D N A s y n t h e s i s in r e c o m b i n a t i o n a l e v e n t s a n d t h e p r o b a b i l i t y of a p h y s i c a l i n t e g r a t i o n of d o n o r D N A into t h e r e c o m b i n a n t s t r u c t u r e m a y be e v a l u a t e d b y c o m p a r i s o n of t h e time-courses of b o t h processes, a n d b y t h e t i m i n g of a p p e a r a n c e of r e c o m b i n a n t s a n d of full resistance t o 82P-decay in r e l a t i o n t o t h e t i m i n g of v e g e t a t i v e r e p l i c a t i o n of t h e m e r o z y g o t e chromosome. Material and Methods Organisms. The strains used were: E. cell K 12: Hfr H, thi-, str 8, tsx 8. E. coli K 12 PA-209: F - , thr-, leu-, the-, trp-, his-, str r, tsx t. Symbols of genes are given according to Taylor and Trotter (1967). Media. Minimal medium M 9 (Adams, 1959), supplemented with 0.2% Casamino acids "Difco" and, when necessary, with amino acids and vitamins, was used for the growth of cultures and for mating experiments. H medium of Stent and Fuerst (1955) with a low content of total phosphate (6 ~.g/ml) was employed for the radioactive experiments. M 9 medium, solidified with agar, enriched appropriately and supplemented with streptomycin (100 ~g/ml), was used for the assay of reeombinants, and nutrient agar for the determination of viable count. Radioactive Techniques. methods employed were similar to those originally described by Fuerst and Stent (1956). 32p-isotope was obtained as carrier free l:[3a2poa from the Instytut Badafi J~drowyeh, Opdi-Swierk, Poland. The 32p was added to H medium to a final specific activity of about 150 mCi/mg P. An overnight culture of Hfr bacteria in "cold" H medium was diluted with 200 parts of the radioactive H medium and incubated for 12 hours. The culture was then diluted (1 : 10) with fresh radioactive H medium and incubated with shaking for a further 3 hours. The labelled bacteria were harvested on Coli-5 membrane filters, washed several times with buffer and resuspended in non-radioactive H medium. This suspension was used for mating with unlabelled F - cells for various periods; the ttfr cells were eliminated with T 6 bacteriophage. The exconjugant F - cells were, directly or after post-incubation, transferred to M 9 medium containing 10% (v/v) glycerol. 0.1 ml samples of such suspension were frozen and stored in liquid nitrogen (-- 196° C). After storage, bacteria were rapidly thawed in a water-bath (43 ° C), diluted in 1~ 9 medium and plated for reeombinants. Mating Procedure. Aliquots of exponentially growing Hfr and 1~-cultures were mixed in the ratio 1:10 and incubated at 37 ° C without shaking. At desired intervals, conjugation was interrupted by addition of T e bacteriophage. In part of experiments, mating mixture after 5 minutes of contact was diluted in the ratio 1:100, and then incubated for appropriate period. This procedure prevents the formation of new active pairs during succeeding mating. Inhibition o] D N A Synthesis. For inhibition of DI~A synthesis, edeine in concentration 350--400 ~g/ml was used. This concentration blocks DNA synthesis completely, instantaneously and reversibly (Piekarowlcz, W~odarczyk and Kunicki-Goldfinger, 1968) without interfering with RNA and protein syntheses (Kury~o-Borowska, 1967). Some more particulars are given in descriptions of the experiments.

Role of DNA Synthesis in Post-Conjugal Genetic Recombination. IX

265

Results DNA

S y n t h e s i s a n d Genetic R e c o m b i n a t i o n

The f r e q u e n c y of r e c o m b i n a t i o n was m e a s u r e d i n d i r e c t l y b y scoring the relative n u m b e r s of T h r + L e u + S t r r a n d T r p + S t r r r e e o m b i n a n t s . F o r assessing the role of D N A synthesis, edeine was a d d e d for various periods to the suspenssion of cells after i n t e r r u p t i o n of m a t i n g a n d e l i m i n a t i o n of I-Ifr cells. B y a d o p t i n g this procedure, a n y effects of edeine on the preceding stages of c o n j u g a t i o n (effective pairs formation, transfer) were excluded. Thus, only the r e c o m b i n a t i o n a l events were influenced. I n one series of experiments, m a t i n g was i n t e r r u p t e d after different times of contact, a n d t h e n edeine was allowed to act for 60 m i n u t e s of p o s t - i n c u b a t i o n . I n a second series, p o s t - i n c u b a t i o n i n the presence of edeine was c o n t i n u e d u n t i l the 1 2 0 t h m i n u t e after the c o m m e n c e m e n t of conjugation, i n d e p e n d e n t l y of t h e d u r a t i o n of m a t i n g . I n b o t h series, samples p o s t - i n c u b a t e d w i t h o u t edeine served as controls. The results of representative e x p e r i m e n t s are shown i n Table 1 a n d 2 a n d i n Fig. 4. I n b o t h cases a m a r k e d decrease i n the n u m b e r of r e c o m b i n a n t s was observed, when D N A synthesis was i n h i b i t e d n o t later t h a n 40---50 m i n u t e s after transfer of the m a r k e r examined. If edeine was added later, its effect was m u c h smaller. Table 1. Relationship between D N A synthesis and genetic recombination. Numbers of recombinants X 103, per ml

Reeombinants

Postincubation

Time of mating (minutes) 10

20

T-L- S T-L-S Trp-S Trp-S

Control Edeine Control Edeine

4 0.6 (15) ---

40 200 11 (27) 70 (35) -2 -0.4 (20)

40

60

80

100

120

300 320 350 500 130 (43) 230 (70) 270 (76) 430 (86) 36 50 70 70 10 (28) 25 (50) 58 (83) 60 (85)

Bacteria were mated for various times as described in "Methods" and then postincubated for 60 minutes in the presence of edeine or without the antibiotic (Control). T-L- S = recombinants Thr+ Leu + Str r; Trp- S ~ T r p + Str ~. Numbers in brackets ~ number of recombinants as per cent of the control. Table 2. Ef]ect of inhibition of D N A synthesis on recombination. Numbers of recombinant8 × 10 ~, per m~

Recombinants

Thr + Leu + Str r Thr+ Leu+ Str r T r p + Str + T r p +Str r

Experimental conditions

Time of conjugation Time of post-incubation 20 40 100 80

70 50

120 0

Control Edeine Control Edeine

3.0 0.52 (17) ---

8.0 4.0 (50) 2.8 0.56 (20)

50.0 50.0 (100) 13.0 12.0 (92)

6.0 1.7 (28) 0.34 0.05 (15)

Bacteria were mated for various time-periods as described in "Methods" and then postincubated without edeine (control) or in presence of edeine till the 120th minute after beginning of mating. Numbers in brackets ~ frequency of recombinants as per cent of the control.

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lYl.Wtodarczyk and W. Kunicki-Goldfinger:

I t seems, that between 40 and 50 minutes after transfer, the recombinational process reached the stage, at which DNA synthesis is not essential.

Development o/Resistance to 3sP-Decay and DNA Synthesis Ability of asp to inactivate genetic determinants, as well as its lethality for bacteria, are almost entirely due to the decay of 8sp atoms in DNA (Fuerst and Stent, 1956). Different donor genetic determinants, when they are already transferred into the recipient cell, are inactivated at approximately the same rate. Moreover, the frequency of unselected markers, when the selected marker is located distally to them, is not affected by 8sP-decay. In other words, the effectiveness of asP-decay is not dependent, in this case, upon its ability to cause fragmentation of the chromosome. I t appears that, concomitantly with penetration of donor DNA into the F - cell, its pairing with homologous segments of the recipient chromosome is induced. The fixed and immobilized labelled donor DNA strand thus becomes resistant to fragmentation due to asP-decay on prolonged mating. Donor markers transferred to F - cells become less and less susceptible to asP-decay, attaining high degree of resistance after ca. 120 minutes of mating. The development of resistance by donor genetic determinants in the recipient cell, found by us (Fig. 1), confirms the results of Jacob and Wollman (1961). The development of resistance to asP-decay does not depend on the time of mating but on the time of succeeding post-incubation (Fig. 1). Variation of the time of mating, from 20 minutes till 120 minutes, has no influence on development of the resistance, on condition that ex-conjugant female cells were postincubated before freezing for appriopriate periods, not shorter than about 50 minutes after transfer of the examined marker. In both cases, the full resistance was, however, not attained. The partial susceptibility to asP-decay, found even after 120 minutes of mating, may be caused by heterogeneity of the examined population. The population of ex-conjugant female cells contains the merozygotes formed during the whole incubation period, during which new active pairs may be formed. The merozygotes, which leter received fragment of the donor chromosome, were incubated for a shorter period, and consequently there was not enough time for development of the resistance. To test this possibility, the experiments were repeated, but the formation of the new active pairs was prevented by dilution of the mating mixture in the ratio 1 : 100 after five minutes of contact. In this case, all merozygotes tested were formed at the same time. The results, shown in Fig. 2, confirmed the assumption. The full, 100 per cent, resistance to 32P-decay was reached already after 70 minutes of mating i.e. 65 minutes after dilution of the mating mixture. The examined markers (Thr, Leu) are transferred after 9 minutes. The time needed for development of the resistance may be then calculated, with fairly good approximation, as 45 to 55 minutes after transfer of markers.

Relationship between Genetic Recombination and Development o/Resistance to a2P-Decay Certainly, the development of resistance to 3sP-decay is not determined by the repair process, since the development of resistance is not observed in Iff_fr cells before the transfer or in endogenous DbTA of F - cells. There was then good reason

Role of DNA Synthesis in Post-Conjugal Genetic Recombination. IX 3

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Fig. I a--c. Inactivstion by 32p-decay of donor genetic determinants in merozygotes and development of their resistance to the decay during post-incubation, a Bacteria were mated for diYferent periods and, after elimination of H]r cells, merozygotes were exposed to 82p-decay at -- 196° C. b Bacteria were mated for different periods; H]r cells were eliminated and merozygotes post-incubated until 120 minutes from the beginning of mating, and then exposed to 32P-decay at -- 196° C. c Effect of inhibition of DNA synthesis on the development of resistance to 3=P-decay. Bacteria were mated for different periods, H]r cells were eliminated and merozygotes postincubated in presence oY edeine until the 120th minute from the beginning of mating, and then exposed to 32P-decay at -- 196° C. The first numbers above the curves denote the time o~ mating, the second ones - - the time of post-incubation. The data were normalized in each case to the zero-dose number of recombinants. A0= 140 mCi/mg P to t h i n k t h a t , the build u p of resistance to 3~P-decay of donor d e t e r m i n a n t s is c o n n e c t e d with the r e c o m b i n a t i o n a l process, t a k i n g place a t the same t i m e i n the F - merozygote. If the effect of i n h i b i t i o n of D N A synthesis on the developm e n t of resistance to 32P-decay shows the same time-course as its effect on the decrease of r e c o m b i n a t i o n frequency, i t follows t h a t the e l i m i n a t i o n of sensitive targets from the r e c o m b i n e d s t r u c t u r e is m e d i a t e d b y D N A synthesis. E x p e r i m e n t s were, therefore, carried out to test the effect of i n h i b i t i o n of D N A synthesis b y edeine on the d e v e l o p m e n t of resistance of donor genetic d e t e r m i n a n t s t o 82P-decay. The labelled H f r cells were m a t e d with u n l a b e l l e d F - cells as described i n "Methods". The c o n j u g a t i o n was i n t e r r u p t e d a n d the I t f r cells were

268

M. Wlodarczyk and W. Kunicki-Goldfinger: 1oo

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l~ig. 2. Development of resistance to 32P-decay of donor genetic determinants transferred into recipient cells. Hit and F - cells were mixed, 5 minutes after mixing diluted 1 : 100, and mated for different periods. HIt cells were then eliminated and merozygotes post-incubated until 120 minutes from the beginning of mating, in presence or absence of edeine. After post-incubation, bacteria were exposed to a2P-decay at -- 196° C. 1 © Mating for 20 minutes; no post-incubation. 2 • Mating for 20 minutes; post-incubationin presence of edeine. 3 ~7 Mating for 20 minutes; post-incubation without edeine. 4-~ Mating for 40 minutes; no postincubation, fi [] Mating for 40 minutes; post-incubation in presence of edeine. 6 × Mating for 40 minutes; post-incubation without edeine. 7 and 8 o, A Mating for 70 and 120 minutes, respectively. There is no influence of post-incubation, independently of presence or absence of edeine 100 8o

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4O 70 Time of mcttin£

120

Fig. 3. Development of resistance to 32P-decay and inhibition of this process by edeine. 1 - - Mating for different periods; elimination of Hit cells; post-incubationuntil 120th minute from the beginning of mating, then exposition to 32P-decay at -- 196° 0 for 15 days. 2 . . . . As above; the only difference - - post-incubation in presence of edeine. Maximal number of recombinants - - number of recombinante after 120 minutes of mating

e l i m i n a t e d after various periods (20, 40 a n d 70 minutes). The suspension of t h e F - merozygotes was t h e n divided i n t o two b a t c h e s ; to the first edeine was added, while t h e second one served as control. The samples of b o t h series were posti n c u b a t e d till the 120th m i n u t e after t h e m i x i n g of m a t e d partners. The aliquots of all i n c u b a t e d samples were stored a t - - 1 9 6 ° C for different time-periods,

Role of DNA Synthesis in Post-Conjugal Genetic Recombination. IX

269

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Fig. 4. Time-course of recombinationprocess and inhibitionof &is process by edeine. 1 - - Mating for different periods; elimination of Hit cells; post-incubation for 60 minutes. 2 . . . . As above; the only difference - - post-incubation in presence of edeine. Maximal number of rccombinants - - number of recombinants after 120 minutes of mating. Remark: At 100th minute - - initiation of the first division of recombinant cells lOO

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Fig. 5. Relationship between effect of edeine on the development of resistance to s2P-decay of donor markers in merozygotes and influence of cdeine on recombination frequency. + decrease of resistance to 3~P-decay after post-incubation in presence of edeine, in per cent of resistance reached after post-incubation without edeine. Bacteria were mated for different periods; H[r cells were eliminated and merozygotes post-incubated in presence or absence of edeine until 120 minutes after beginning of mating. The bacteria were then exposed to 32P-decay at -- 196° C for 10 days. Mating mixture was not diluted; due to concomitant pair formation during mating - - full resistance, in this case, was not attained. Q, O decrease of recombination frequency after post-incubation in presence of edeine, in per cent of recombination frequency reached after post-incubation without edcine. G Mating for different periods; post-incubation in presence of edeine until 120 minutes from the beginning of mating. O Mating for different periods; post-incubation in presence of edeine for 60 minutes after mating

t h a w e d a n d tested as described i n "Methods". R e p r e s e n t a t i v e results are g i v e n in Fig. 1 middle f i g u r e - c o n t r o l , a n d Fig. 1, lower f i g u r e - i n h i b i t i o n of D N A synthesis. The curves i l l u s t r a t i n g i n h i b i t i o n b y edeine of r e c o m b i n a t i o n process, m e a s u r e d b y r e c o m b i n a n t s frequencies (Fig. 4), a n d of d e v e l o p m e n t of t h e resistance to 32P-decay (Fig. 3), are quite similar. The relationship b e t w e e n the t i m e of edeine action a n d the per cent decrease of r e c o m b i n a t i o n frequency, or per cent i n h i b i t i o n of d e v e l o p m e n t of t h e resistance to 32P-decay, is i d e n t i c a l (Fig. 5).

270

hi. Wiodarczyk and W. Kunicki-Goldfinger:

The time-limits of the development of resistance of donor determinants to 82P-decay are thus almost identical with the time-course of the recombinational process. Discussion The experiments reported in this paper were done to test a hypothesis about the mutual interrelationship between genetic recombination and the development of resistance of donor genetic determinants to 8~P-decay. I t appears that both processes are mediated b y some common factor or factors, susceptible to inhibition of DNA synthesis. Evidence that recombination in E. coli occurs in the absence of DNA replication does not contradict our results. In DNA mutants, used by Joshi and Siddiqi (1968), it was only normal, physiological replication which had been blocked, while repair-replication and phage-DNA syntheses were unaffected. Edeine, used in the present study, seems to inhibit all, or at least most types of DNA synthesis in the cell. The DNA synthesis, which is indispensable for recombination, may be identical with repair-synthesis or may be a process specific for recombination. The assumption that, the structure resistant to a~P-decay is formed before and independently of the normal vegetative replication of the recombinant chromosome in the F - ex-conjugant cells, is supported by following observations. This report shows that 45 to 55 minutes after transfer of the male genetic determinant, the recombinagonal process is unaffected by inhibition of DNA synthesis and the resistance to 32P-decay is fully evolved. This is in fairly good accordance with the beginning of activation of specific enzyme proteins, due to formation of recombinant structure (Pritchard, 1965; Joshi and Siddiqi, 1968). The formation of this structure is not influenced by inhibition of the vegetative replication of ex-conjugant chromosome (the same authors). I t may be than presumed that, the process is independent of vegetative replication of chromosome. The formation of the recombinant structure and of structure resistant to 3~P-decay is completed before the beginning of replication of recombinant chromosome. According to Tomizawa (1960) ca. 100 minutes are required after mating begins, before there is any increase in the number of recombinant structures. I t is in agreement with the results of Wollman, Jacob and Hayes (1956), Hayes (1957), and many later reports. In these investigations, the recombinant cells started to divide 100 minutes after beginning of mating (Fig. 4). Taking into account the time relation be tween the cell division and the chromosome replication (Abe and Tomizawa, 1967; Clark and Maaloe, 1967; Cooper and Itelmstetter, 1968; ttelmstetter, 1967; Helmstetter and Cooper, 1968 ; Myeielski, Lityfiska and Knnicld- Goldfinger, 1968; Mycielski et al., 1969) it may be concluded that vegetative replication of the exconjugant chromosome certainly starts less than 50 minutes before the beginning of cell division, i.e. later than 50 minutes after commencement of mating. The results cited, as well as those reported by Jacob and Wollman (1961), and Gross (1963), suggest that recombinant structure is complete within 50 to 70 minutes after transfer. The results presented in this paper show that the structure resistant to s~P-deeay is formed within the same period. They show also tha~ a time-course of both processes, recombination and development of resistance, is almost identical. The conclusion that both processes are mediated by a common

Role of DNA Synthesis in Post-Conjugal Genetic Recombination. IX

271

event, d e p e n d i n g on u n a b a t e d D N A synthesis, seems t h e n to be quite well subs t a n t i a t e d . The f o r m a t i o n of t h e s t r u c t u r e r e s i s t a n t to 32p-decay is t h u s due n o t to preceding r e p l i c a t i o n of r e c o m b i n a n t molecule, b u t is going on c o n c o m i t a n t l y w i t h r e c o m b i n a t i o n a l e v e n t s a n d is c o m p l e t e d before beginning of t h e v e g e t a t i v e replication. This conclusion is i n c o m p a t i b l e w i t h t h e p h y s i c a l presence in t h e r e c o m b i n a n t molecule of a r e l a t i v e l y big s t r e t c h of exogenous s t r a n d labelled w i t h r a d i o a c t i v e phosphorus. The i n a c t i v a t i n g effect of a2p-decay, even when o n l y one s t r a n d is labelled w o u l d be s t r o n g enough to be a p p a r e n t (see e.g. Bressler a n d L a n z o v , 1967; Bressler, L a n z o v a n d L u k j a n i e c - B l i n k o v a , 1968). To a c c o u n t for t h i s fact, as well as for m a n y r o u n d s of r e c o m b i n a t i o n o b s e r v e d in H a y e s t y p e of H f r (Lederberg, 1957; Anderson, 1958; Bressler, L a n z o v a n d Blinkova, 1967; W o o d , 1967), a n y r e c o m b i n a t i o n m o d e l assuming p h y s i c a l i n t e g r a t i o n of donor D N A i n t o t h e r e c o m b i n a n t molecule seems u n s u i t a b l e . Thanks are due to Dr. H. Ptuciennik from the Radiochemistry Department, Warsaw University, ~or the facilities to carry out a part of this work, and to Professor Dr. E. Borowski for providing edeine. We are also indebted to the Commission of General Microbiology, the Polish Academy of Sciences, for financial support.

References

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Role of DNA Synthesis in Post-Conjugal Genetic Recombination. IX

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